INDEX

An Immunohistochemical Study of LH-RH and S-100 Protein in the Hypothalamus and the Pars Tuberalis of the Anterior Pituitary Gland

Takashi Yanagisono, Jun Kurita, Hiroyuki Kimura, Morihiro Yoshida and Eisuke Sakuma
The First Department of Anatomy, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan

Running Title: Immunohistochemistry of the pars tuberalis
Key Words: Rat, Anterior pituitary, Pars tuberalis, LH-RH, S-100 protein
Address for correspondence: Takashi Yanagisono, The First Department of Anatomy, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
Tel (+81) 52-853-8121; Fax (+81) 52-842-3210;

SUMMARY
The distribution of nerve fibers containing luteinizing hormone-releasing hormone (LH-RH) within the median eminence was demonstrated in the 1970é’s and 1980é’s. A few LH-RH containing fibers have been also detected in the pars tuberalis (Naik 1975a). The role of these fibers in the pars tuberalis have not been fully elucidated.
Adult Wistar-Imamichi male rats were separated into two groups for immunohistochemical study about LH-RH and S100 protein (S-100) and electron microscopy. Specimens including the hypothalamus and the pituitary gland were prepared for the immunohistochemical light microscopic observations, and sagittal serial mirror sections with 5 um thickness were used. Each mirror pair was stained by using antibodies towards LH-RH and S-100 respectively. Sections were obtained from the region 300 um to both sides from the sagittal center. Many LH-RH positive fibers were observed both in the primary plexus and in the hypothalamus that corresponded to the caudal half of the portal vein area of the pars tuberalis. The reaction products for LH-RH were also observed as thin lines with several varicosities in the caudal half of the portal vein area of the pars tuberalis. LH-RH reactions were also observed around the cells that were S-100 positive on their mirror sections. Several cells showing a weak reaction to S-100 were scattered in the pars tuberalis at the approximately center position, while the strong reactions were observed as clusters on 100-300 um lateral from the sagittal center.
It was strongly suggested that LH-RH information was transmitted to the folliculo-stellate cells in the pars tuberalis and the interconnected folliculo-stellate cells transported some signals to modulate LH release from the LH cells within the pars distalis.

INTRODUCTION
There are numerous reports about the localization, morphological characteristics, gene expression and regulation of the gonadal-pituitary axis of LH-RH and LH-RH neurons (1-12). Detailed description of the distribution of LH-RH positive nerve fibers in the periventricular area including the median eminence was made in the 1970é’s and 1980é’s (1, 2, 3, 10, 13, 14). Naik (2) performed immunohistochemical studies using fluorescent histochemical technique, and pointed out that a few LH-RH containing nerve fibers were found in the pars tuberalis, but he did not clarify the significance of these fibers. The pars tuberalis has recently gained attention for its role in the photoperiodic regulation of hormone secretion in the anterior pituitary gland; especially about prolactin and gonadotropin (15, 16 17, 18). These are commonly recognized to be related with the melatonin receptors on the cells in the pars tuberalis.
In the earlier stages of electron microscopic research on the rat anterior pituitary gland, Farquhar (19) reported the agranular cells arranged around a follicle to be adrenocorticotropic hormone (ACTH)-secreting cells. Based on the existence of S-100 protein in the folliculo-stellate cells, Nakajima et al. (20) suggested that they were originated from the neuro-ectodermal cells. The existence of cell-to-cell communication within the organ has mainly been described between the folliculo-stellate cells in the monkey, rat and teleost anterior pituitary glands (21, 22, 23, 24, 25, 26). Moreover, Soji and his coworkers performed the experiments about the formation of gap junctions in the rat anterior pituitary gland under several physiological conditions, and pointed out their possible role in the conduction of information through a system of interconnecting follicles and folliculo-stellate cells (25, 26, 27).
In this study, the authors demonstrate another regulatory mechanism of hormone secretion in the anterior pituitary gland.

MATERIALS AND METHODS
A total of 20 Wistar-Imamichi male rats were used. Ten were 50 day-old and 10 were 60 day-old. Five of 50 day-old and 5 of 60 day-old rats were used for immunohistochemistry and other 5 animals in each groupe were used for electron microscopy. All animal experiments were performed under é“The Guidelines for Animal Experimentationé” of the Experimental Animal Science Center of the University. All animals were deeply anesthetized with Nembutal® (pentobarbital), and the specimens including the hypothalamus and the pituitary gland were obtained from each rat.
For the immunohistochemical light microscopic observation, the median eminence areas with pituitary gland were fixed overnight in Bouin's solution without acetic acid at 4°C, dehydrated in an ethanol series, and embedded in Paraplast embedding media (Sigma Chem. Co, St. Louis, MO, USA). Sections were obtained at the region from the sagittal center to 300 um on both sides, and sagittal serial mirror sections with 5 um thickness were used. Serial mirror image sections were mounted on poly-L-lysine-coated slide glasses, then used for immunohistochemistry. Each pair of mirror sections were immunostained respectively for LH-RH and S-100 protein (S-100) by the peroxidase-labeled antibody method of Nakane and Pierce (28) with a slight modification (29). The LH-RH immunostained specimens were slightly counter-stained by Mayeré’s hematoxylin. The antiserum against LH-RH (Anti-mammal LH-RH rabbit serum: Biogenesis Ltd., Poole, UK (name changed from UCB-Bioproduct)) and S-100 (anti-bovine S-100 alpha subunit rabbit serum: Life Science Lab., Tokyo, Japan) were commercially obtained (Fig. 1). These sera were diluted 1:20,000 and 1: 8,000, respectively. At the sagittal center (SC) section, specimens were checked as to whether the sectioning was parallel or not to the anatomical SC plane, and the accurately parallel specimens were used for observations. The specimens which were sliced obliquely were not used.
The electron microscopical observation was performed to confirm the immunohistochemical results. Each five animals (50- and 60 day-old male rats) were fixed by perfusion from the left ventricles of the hart for 10 min. The perfused fixative contained 2.5% glutaraldehyde and 2% sucrose buffered with 0.05M sodium cacodylate (pH 7.4). After the perfusion, the pars tuberalis (pituitary glands) with hypothalamus were removed from the animals and cut into three approximately as explained in figure 2. After two hours postfixation in 1% osmium tetroxide fixative, the tissues were embedded in epoxy resin. Thin sections were prepared parallel with sagittal plain. Sections were obtained at 4 plains as approximately SC plain, 100um from SC (100um-SC), 200um from SC (200um-SC) and 300 um from SC (300um-SC) plain and set on 50 or 75 copper grids. The all specimens were stained with uranyl acetate and lead citrate and then observed using a Hitachi H-500 and H-7000 transmission electron microscope.
For convenience of explanation, the hypothalamo-tuberal portion was tentatively named as follows. A summary of the abbreviations is shown in figure 1. The pars tuberalis was separated into three portions from cephalad part to caudal part as: the primary plexus portion (PP), anterior portion of the portal vein segment (APV) and posterior portion of the portal vein segment (PPV). Similarly, the floor of the third ventricle was separated into three parts as: the primary plexus portion of the median eminence (PPM), anterior portion of the portal vein segment of the median eminence (APVM) and the posterior portion of the portal vein segment of the median eminence (PPVM). A small protuberance indicated by the arrow was named the transitional zone of the hypothalamus and infundibular recess (THI). A bend of the floor that corresponded to the THI was named the bend of the floor of the third ventricle (BFT). The region indicated by arrowheads is expressed as "bottom of the floor" in this text (Fig. 2-2).

RESULTS
No significant difference was found between 50 and 60 day-old rats. Each serial sections of sagittal plain which intervals were 100um showed distinct changes about courses and localization of LH-RH positive fibers.

A) Immunohistochemistry of LH-RH and S-100 protein
1) Sagittal center plane
The PP of the pituitary gland was not observed in the sagittal center (SC) plane. A few capillaries were found on and in the é“bottom of the flooré” of the third ventricle instead. A few anti-LH-RH reaction products were observed as fine lines and numerous dotted lines on the front half of the floor of the third ventricle. Although most dotted lines of deposits ran through to the PPVM just below the third ventricle, a few arrays of deposits of the reaction ran down to the capillaries at the é“bottom of the flooré”. At APVM and PPVM, a few deposits were observed as dotted lines just below the third ventricle and almost all lines of deposits showed direction to the infundibular stalk. A few reaction end products were also observed at THI and a few lines of the deposits were observed from the THI to the infundibular stalk (Fig. 3L). Blood vessels were clearly observed between the brain tissue and the pars nervosa and vessels entering brain tissue were observed (Fig. 2-1). Contrary to this, no blood vessels and no pars tuberalis areas were observed in the bend of the floor of the third ventricle.
Anti-S-100 reactions in the brain in the SC plane were observed as usual. The reaction end products were mainly observed on the glia cells and their cell processes. Whereas almost all of the processes crossed the floor and reached the bottom of the floor of the third ventricle, no processes traversed to the pituitary section such as the APV or PPV (Fig. 3S). On the other hand, only cells weakly positive for S-100 protein were observed in the PPV (Fig. 3S).

2) One hundred micrometers from the SC (100um-SC)
At 100um-SC, a few primary plexus with short PP were attached to the é“bottom of the flooré” (Fig. 4L). It was clearly observed that the arrays of dots of anti-LH-RH reaction products ran to the plexus at PP. Although the primary plexus already joined to portal veins, a few arrays of the dots ran toward PPV. Dots of reaction of LH-RH in the THI were increased (Fig. 4L). Though the BFT area was avascular, LH-RH positive dots were numerous and the array seemed to come from the paraventricular area (Fig. 4L). At this plane, no pars tuberalis area was observed between the infundibular stalk and the hypothalamus indicated as BFT in figure 2.
At 100um-SC, the third ventricle was narrowed dorsally. Clusters of densely S-100 positive cells were observed in the PPV area. The clusters showing the apical cell processes were clearly observed (Fig. 4S). In the anterior pituitary, near the junction between the anterior lobe and the pars tuberalis, the population of S-100 positive cells was mildly increased.

3) Two hundred micrometers from the SC (200um-SC)
At 200um-SC, PP was lengthened and numerous primary plexuses were observed under the é“bottom of the flooré” (Fig. 5L-1). The arrays of dots of anti LH-RH reaction products running to the plexus at PP area increased drastically in this area. It was very interesting that an area of little or no reaction was observed at APVM in this plane. It was often observed on PPVM at this plane that the é“bottom of the flooré” faced PPV between the portal veins and sometime touched with PPV (Fig. 5L-2). The interval between the pars tuberalis and the é“bottom of the flooré” was clearly observed in other portions (such as PP) (Fig. 5L-3). Blood vessels were found in the THI area and numerous LH-RH positive dots were found near the BFT (Fig. 5L-1). A few slender LH-RH positive fibers with small varicosities were observed in the pars tuberalis and then disappeared around the tuberal cells in the PPV (Figs. 5L-3 and -4). Most of the cells were S-100 positive cells by immunohistochemistry. This observation was also found in mirror sections of 100um-SC and 300um from the SC (300um-SC), but were scarce.
Anti-S-100 reactions in the brain on 200um-SC were observed as usual. Only the basal part of the third ventricle was observed. While only few S-100 positive cells existed on the APV, numerous clusters of densely S-100 positive cells were observed in the PPV area (Fig. 5S-1,2). The clusters showing the apical cell processes were also clearly observed (Fig. 5S-2). A small area of the pars tuberalis was observed within the BFT.

4) Three hundred micrometers from the SC (300um-SC)
Only the margin of the third ventricle was observed at this plane (300um-SC). The primary plexus was also not observed and only PP fragments were observed under the é“bottom of the flooré” (Fig. 6L). While the PP and the primary plexus were no longer visible, the LH-RH deposits were widely observed (Fig. 6L). At the PPVM, it was easy to find the array arrangement to the PPV. At this plane, the pars tuberalis was observed within the BFT (Fig. 6L).
Anti S-100 reactions in the brain 300 um-SC were observed as usual. Numerous clusters of densely S-100 positive cells were still observed in the PPV area. The cluster of cells showed fine and intertwining cell processes (Fig. 6S).

B) Immunohistochemistry of LH-RH and S-100 protein on mirror sections
It was interesting to note on the mirror sections of the anti-LH-RH and S-100 studies that small round deposits of LH-RH reaction were observed around some S-100 positive cells in the PPV 200um-SC (Figs. 7L and S). The cell bodies were also positive for LH-RH (Figs. 7L and S). A few slender LH-RH positive fibers with small varicosities were observed in the pars tuberalis and then disappeared around S-100 positive cells in the PPV (Figs. 5L-3 and –4 and 7L and 7S). This observation was also found in mirror sections of 100um- and 300 um-SC, but were scarce.

C) Electron microscopy
Following observations were most obvious at 200um-SC, bot also observed at 100um-SC and 300um-SC. At PPM, numerous nerve fibers were found on the upper side (brain side) of the capillaries. In the nerve fibers, secretory granules (80-120 nm in diameter) and small vesicles (50-80 nm in diameter) were stuffed. The other side of the capillaries faced to glandular or epithelial cells of pars tuberalis. The pituitary gland and the brain were clearly separated by basement membrane of capillaries or some fibroblastic cells in this portion. At APV and APVM, similar observations as PP were obtained. The capillaries were occasionally observed between the brain and pars tuberalis (Fig. 8-1). At APV, granulated cells contained small granules (100-150 nm in diameter) were observed between the agranulated cells (Fig. 8-1). Clusters of the agranulated cells that arranged around the follicle lumen were observed. The agranulated cells projected microvilli and cilia into the lumen (Fig. 8-1). Some of the floor of the third ventricle protruded toward PPV of the pars tuberalis (Fig. 8-1).
The nerve fiber bundle with dense granules, which diameter was 100 nm, were invaded into the pars tuberalis and contacted to the agranulated cells without the basement membrane (Fig. 8-1). At the near of the site, a small cell process contacted to an agranulated cell (Fig 8-2). The small cell process contained numerous small granules (80-110 nm in diameter) and vesicles (50-100 nm in diameter). Concerning to this diameter and density, the granules corresponded to granules in nerve fibers in the median eminence (Fig 8-1), whose granules were considered as neuroendocrine granules. Agranulated cells contained cell fragments which contained numerous dense cored granules, small vesicles and mitochondria (Fig. 8-2). The size of the fragment showed good agreement to round deposits of LH-RH reactions. Although, both cell and the fragment were clearly separated by two layers of plasma membranes of both agranulated cell and fragment, no basement membrane was observed between the cell space (Fig. 8-2). The agranulated cells arranged around the small follicle (double arrows in Figs. 8-1 and 8-2).

DISCUSSION
This study demonstrated that the neuroendocrine fibers terminated not only in the primary plexus region (PP in Fig. 2) but also within the caudal part of the pars tuberalis (PPV in Fig. 2) of the pituitary gland. In addition, immuno-histochemically LH-RH positive clusters existed around the S-100 positive cells in the pars tuberalis.
Although there are numerous reports about the localization, morphological characteristics, gene expression, and regulation of the gonadal-pituitary axis of LH-RH and LH-RH neurons (1-12, 30), only few reports have referred to the existence of LH-RH positive nerves in the pars tuberalis (2). A detail of the distribution of the LH-RH positive nerve fibers in the periventricular area including the median eminence was demonstrated in the 1970é’s and 1980é’s (1, 2, 3, 4, 10, 13, 14). According to Naik (2) and Foster and Youngai (10), the LH-RH positive nerve fibers mainly derived from the preoptic and prechiasmatic area, arcuate nuclei and ventrolateral-premammillary body in rats and rabbits. In the present study, the LH-RH positive nerve fibers running down to the é“bottom of the flooré” in the cephalad portion of the third ventricle floor (PPM) were mainly derived from the nucleus situated in the cephalad part of the hypothalamus. These fibers terminated mainly on the primary plexus. Contrarily, the fibers running down to the é“bottom of the flooré” in the caudal portion of the floor of the third ventricle (PPVM) were derived from the nucleus in the ventrolateral-premammillary body. The LH-RH fibers were supplied from both the cephalad and the caudal parts of the hypothalamus. In addition, there were a few LH-RH fibers to the é“bottom of the flooré” in the middle part indicated as APVM in figure 1.
Upon reconstruction of a frontal plane view from the total of sagittal sections, the distribution pattern of the LH-RH positive nerve fibers showed a specific pattern of localization. While the fibers were numerous in zones 200 um lateral from the SC, only a few fibers were found in the very central area of the hypothalamus. On the other hand, the PP and primary plexus were clearly observed in zones 200 um lateral from the SC, while few PP and primary plexus were observed in zones 100 um lateral from the SC. The primary plexus and pars tuberalis expanded with distance from the SC up to 200 um, then rapidly decreased. These distribution patterns of the LH-RH positive fibers and primary plexuses were reasonable with regard to the architecture of the hypothalamic nuclei in the hypothalamus, and the high density of hypophyseal arteries in the lateral sides of infundibular stem.
In the earlier stages of electron microscopic research on the rat anterior pituitary gland, Farquhar (19) reported the agranular cells arranged around a follicle to be adrenocorticotropic hormone (ACTH) secreting cells. Subsequently, chromophobic cells surrounding the follicle were named "folliculo-stellate cells" based on their morphological characteristics, and were proven to have cytoplasmic contents including S-100 protein (20) and ß-adrenergic receptors (31). Based on the existence of S-100 protein in the folliculo-stellate cells, Nakajima et al. (20) supposed that they had the neuro-ectodermal origin. It has been generally accepted that they are not adrenocorticotropic cells but a distinct cell type.
The pars tuberalis has recently gained attention for its role in the photoperiodic regulation of hormone secretion in the anterior pituitary gland; especially prolactin and gonadotropin (15-18). These are commonly recognized to be related with the melatonin receptors on the cells in the pars tuberalis.
According to Morgan et al. (15), few follicular cells were observed in the ovine pars tuberalis. In our observation on the pars tuberalis of the rat pituitary gland, S-100 positive cells were clearly seen in zones 100-300 um lateral from the center of the pars tuberalis. It has been widely accepted that S-100 is the marker protein of the folliculo-stellate cells in the rat (Nakajima et al., 1980). This could be observed only in the primary plexus region in this study.
In the present study, the existence of LH-RH reactions around the S-100 positive cells in the pars tuberalis was clearly demonstrated by immunohistochemical studies of mirror sections. It is strongly suggested that the LH-RH was incorporated into the S-100 positive cells. The phenomenon of non-synaptic release of Gn-RH was reported in the fresh fish brain from immuno-electronmicroscopy studies (30). According to these, Gn-RH was released non-synaptically from dense cored vesicle (DCV) containing fiber varicosities and it exerted its modulatory action on Gn-RH receptors located on nearby as well as distant target neurons. The present study demonstrated that the neuroendocrine fibers and their varicosities were found in the pars tuberalis and nearby agranular cells. It was strongly suggested that an intimate relationship existed between the agranular cells and the LH-RH containing neurons. The possibility of a certain photoperiodic regulation of the anterior pituitary hormones through LH-RH neurons to folliculo-stellate cells has been proposed by many investigators. We believe there is a more positive action between the LH-RH neurons and the folliculo-stellate cells in the pars-tuberalis and the hormone regulating process in the pars-distalis.
Though the gap junctional connections within the anterior pituitary gland are considered to be stable, the gap junctional connections between agranular cells in the pars tuberalis may not be so stable (32). We speculated that the "message" of LH-RH could be transmitted by the network system of interconnected folliculo-stellate cells through gap junctions.
The anterior lobe of the rat pituitary gland contains few nerve fibers (33-36). The avenues for LH-RH to reach the anterior lobe from posterior or intermediate lobes are remote (37) since there are only few direct anatomical connections (38, 39). Thus, a more rapid conduction system must be employed such as interconnected folliculo-stellate cells from the pars tuberalis to the pars distalis. Our previous studies showed that gap junctional connections existed only between folliculo-stellate cells in case of adult rats (25). No gap junctions were observed before day 20 but once they appeared, the density of gap junctions in the gland increased with development until the time the animals (Wistar-Imamichi rats) had fully matured, by approximately 45 days (27). Thereafter, gap junction numbers were influenced by the concentration of sex steroids, which fluctuates with the development of the reproductive axis, estrous cycle, pregnancy and ovariectomy/castration (40-42). The findings from our previous reports indicate that the agranular folliculo-stellate cells play an important role in the pituitary gland, perhaps by participation in the regulation of hormone secretion.
It can be estimated that each folliculo-stellate cell participates in the formation of three gap junctions. If information enters the cell from one of the three gap junctions and this information is transported to neighboring cells via the other two gap junctions, the number of folliculo-stellate cells possessing the information can be raised exponentially. When this information conduction is achieved twenty times, the total number of the cells with the information reaches about one million. One million is almost equal to the total numbers of the cells composing the whole anterior pituitary gland of a rat (25). Gap junctions are composed of transmembrane channels allowing the free transcellular exchange of small cytoplasmic molecules such as Ca2+ ions. The cell-to-cell passage of Ca2+ ion can produce Ca2+ signalling observed within pituitary glands (43, 44).
When one folliculo-stellate cell in the pars tuberalis catches the information from LH-RH, this information will be easily transmitted throughout the anterior pituitary gland via gap junctions existing between folliculo-stellate cells. Moreover the "message" is conducted to target cells such as gonadotrophs by paracrine effects from the folliculo-stellate cells (45, 46). Thus we could observe discharged gonadotropes and stuffed gonadotrophs, as well as the status of their secreting cycle.

ACKNOWLEDGEMENTS
The author gives great thanks to Professor Damon C. Herbert (The Health Science Center at San Antonio, University of Texas), Dr. Nobuyuki Shirasawa (Department of Anatomy. Wakayama Medical Collage) and Professor Tsuyoshi Soji (The First Department of Anatomy, Nagoya City University, Medical School) for their support and advice during the course of this study.

REFERENCES
1. Barry, J, and B Carette 1975. Immunofluorescence study of LRF neurons in primates. Cell Tissue Res. 164(2): 163-178
2. Naik, D.V. 1975a. Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescent microscopy. Cell Tissue Res. 157(4): 423-436
3. Naik, D.V. 1975b. Immuno-electron microscopic localization of luteinizing hormone-releasing hormone in the arcuate and median eminence of the rat. Cell Tissue Res. 157(4) 437-455
4. Naik, D.V. 1976. Immuno-histochemical localization of LH-RH during different phases of estrus cycle of rat, with reference to the preoptic and arcuate neurons, and the ependymal cells. Cell Tissue Res. 173(2): 143-166
5.Setalo, G., S. Vigh, A.V. Schally, A. Arimura, and B. Flerko 1975. LH-RH-containing neural elements in the rat hypothalamus. Endocrinology 96(1): 135-142
6. Siverman, A.J. and L.C. Krey 1978. The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections. Brain Res. 157(2): 233-246
7. Ibata, Y., K. Watanabe, H. Kinoshita, S. Kubo, Y. Sano, E. Hashimura, and K. Imagawa 1979. The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence. Experimental immunohistochemistry and radioimmunoassay. Cell Tissue Res. 189(3): 381-395
8.Rethelyi, M, V.S. Setalo, I. Merchenthaler, B. Flerko, and P. Petrusz 1981. The luteinizing hormone releasing hormone-containing pathways and their co-termination with tanycyte processes in and around the median eminence and pituitary stalk of the rat. Acta Morphol. Acad. Sci. Hung. 29(2-3): 259-283
9. Kelly, M.J., T.P. Condon, J.E. Levine, and O.K. Ronnekleiv 1985. Combined electrophysiological, immunocytochemical and peptide release measurements in the hypothalamic slice. Brain Res. 345(2): 264-270
10. Foster, W.G. and E.V. Youngai 1991. An immunohistochemical study of the GnRH neuron morphology and topography in the adult female rabbit hypothalamus. Am. J. Anat. 191(3): 293-300
11. Nakazawa, K., U. Marubayashi and S.M. McCann 1991. Mediation of the short-loop negative feedback of luteinizing hormone (LH) on LH-releasing hormone release by melatonin-induced inhibition of LH release from the pars tuberalis. Proc. Natl. Acad. Sci. USA 88(17): 7576-7579
12. Dé’Este, L., H. Kulaksiz, U. Rausch, R. Vaccaro, T. Wenger, Y. Tokunaga, T.G. Renda, and Y. Cetin 2000. Expression of guanylin in é“pars tuberalis-specific cellsé” and gonadotrophs of rat adenohypophysis. Proc. Natl. Acad. Sci. USA 97(3): 1131-1136
13. Baker, B.L., W.C. Dermody, and J.R. Reel 1975. Distribution of gonadotropin-releasing hormone in the rat brain as observed. Endocrinology 97: 125-135
14. Baker, B.L., F.J. Karsch, D.L. Hoffman, and W.C. Beckman Jr. 1977. The presence of gonadotropic and thyrotropic cells in the pituitary pars tuberalis of the monkey (Macaca mullata). Biol. Reprod. 17: 232-240.
15. Morgan, P.J., T.P. King, W. Lawson, D. Slater, and G. Davidson 1991. Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis. Cell Tissue Res. 263(3): 529-534
16. Morgan, P.J. and L.M. Williams 1996. The pars tuberalis of the pituitary: a gateway for neuroendocrine output. Rev Reprod. 1(3): 153-161
17. Barett, P., M. Morris, W.S. Choi, A. Ross, and P.J. Morgan 1999. Melatonin receptors and signal transduction mechanisms. Biol. Signals Recept. 8(1-2): 6-14
18. Morgan, P.J. 2000. The pars tuberalis: the missing link in the photoperiodic regulation of the prolactin secretion? J. Neuroendocrinol. 12(4): 287-295
19. Farquhar, M.G. 1957. "Corticotrophs" of the rat adenohypophysis as revealed by electron microscopy. Anat. Rec., 127: 291.
20. Nakajima, T., H. Yamaguchi, and K. Takahashi 1980. S100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res., 191: 523-531
21. Fletcher, W.H., N.C. Anderson, and J.W. Everett 1975. Intercellular communication in the rat anterior pituitary gland: An in vivo and in vitro study. J. Cell Biol. 67: 469-476.
22. Abraham, M., C. Sandri, and K. Abert 1979. Freeze-etch study of the teleostean pituitary. Cell Tissue Res., 199: 397-407.
23. Herbert, D.C. 1980. Intercellular junctions in the rhesus monkey pars distalis. Anat. Rec. 195: 1-6.
24. Wilfinger, W.W., W.J. Larsen, T.R. Downs, and D.L. Wilber 1984. An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells. Tissue Cell. 16: 483 - 497.
25. Soji, T., and D.C. Herbert 1989. Intercellular communication within the rat anterior pituitary gland. Anat. Rec. 224: 523-533.
26. Soji, T., and D.C. Herbert 1990. Intercellular communication within the rat anterior pituitary gland. II. Castration effects and changes after injection of luteinizing hormone-releasing hormone (LH-RH) or testosterone. Anat. Rec. 226: 342-346.
27. Soji, T., T. Yashiro, and D.C. Herbert 1990. Intercellular communication within the rat anterior pituitary gland. I. Postnatal development and changes after injection of luteinizing hormone-releasing hormone (LH-RH) or testosterone. Anat. Rec. 226: 337-341.
28. Nakane, P.K., and G.B. Pierce 1967. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J. Cell Biol. 33: 307-318
29. Shirasawa, N., F. Yoshimura, E. Miyashita, T. Yashiro, Y. Sumi, and T. Suzuki. 1983. Quantification of immunohistochemical model sections. Cell Mol. Biol. 29: 327-329
30. Oka, Y, and M. Ichikawa 1992. Ultrastructural characterization of gonadotropin-releasing hormone (Gn-RH)-immunoreactive terminal nerve cells in the dwarf gourami. Neuroscience Letters. 140: 200-202
31. Findell, P.R., and Weiner, R.I. 1988 Bovine pituitary folliculo-stellate cells have beta-sdrenergic receptore positively coupled to adrenosine 3é”, 5é”- cyclic monophosphate production. Endocrinology 123(5) 2454-2461
32. Nishizono, H., T. Soji and D.C. Herbert 1993. Intercellular communication within the rat anterior pituitary gland. V. Changes in cell-to-cell communications as a function of timing of castration in male rats. Anat. Rec. 235: 577-582.
33. Shioda, T., and N. Iwakawa 1992. Probable sensory innervation of the rat anterior pituitary. Histochemical J. 24: 461
34. Jacobson, G., and B. Meister 1996. Molecular components of the exocytotic machinery in the rat pituitary gland. Endocrinology, 137: 5344-5356.
35. Lu, C.R., F.T. Ming, L.I. Benowitz and G. Ju 1995. Evidence for axonal sprouting in the anterior pituitary following adrenalectomy in the rat. J. Endocrinol. 147: 161-166.
36. Liu, Y., J.F. Morris, and G. Ju 1996. Synaptic relationship of substance P-like-immunoreactive nerve fibers with gland cells of the anterior pituitary in the rat. Cell Tissue Res. 285: 227-234.
37. Asa, S.L., K. Kovacs, and S. Melmed 1995. The hypothalamic-pituitary axis. In: The Pituitary, S. Melmed, ed. Blackwell Science, Cambridge, pp. 3-44.
38. Théret, C, and E. Tambois 1963. Étude ultrastructurale des rapport expérimentoux entre des cellules alpha et des fibers neurovégétative dans l'adénohypophyse du rat. Ann. Endocrinol. 24: 421-440.
39. Kurosumi, K., and Y. Kobayashi 1980. Nerve fibers and terminals in the rat anterior pituitary gland as revealed by electron microscopy. Arch. Histol. Jap. 43: 141-155.
40. Soji, T., H. Nishizono, T. Yashiro, and D.C. Herbert 1991. Intercellular communication within the rat anterior pituitary gland. III. Postnatal development and periodic changes of cell-to-cell communications in female rats. Anat. Rec. 231: 351-357.
41. Soji, T., T. Yashiro, and D.C. Herbert 1992. Intercellular communication within the rat anterior pituitary gland. IV. Changes in cell-to-cell communications during pregnancy. Anat. Rec. 233: 97-102.
42. Kurono, C. 1996. Intercellular communication within the rat anterior pituitary gland. VI. Development of gap junctions between folliculo-stellate cells under the influence of ovariectomy and sex steroids in the female rat. Anat. Rec. 244: 366-373.
43. Xiong, Z, and G.R. Strichartz 1998. Inhibition by local anesthetic of Ca2+ channels in rat anterior pituitary cells. Eur. J. Pharmacol. 363(1): 81-90
44. Petit, A., C. Bleicher, and B.T. Lussier 1999. Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotroph. Can J. Physiol. Phamacol. 77(7): 520-528
45. Bilezikijan, L.M., A.Z. Corrigan, A.L. Blount, and W.W. Vale 1996. Pituitary follistatin and inhibin subunit messenger ribonucleic acid levels are differentially regulated by local and hormonal factors. Endocrinology 137(10): 4277-4284
46. Bilezikijan, L.M., A.V. Tyrnbull, A.Z. Corrigan, A.L. Blount, and W.W. Vale 1998. Interleukin-1beta regulates pituitary follistatin and inhibin/activin betaB mRNA levels and attenuates FSH secretion in response to activin-A. Endocrinology 139(7): 3361-3364

Figure Legends
Figure 1: S-100 protein positive cells in the anterior pituitary gland. Numerous positive cells and cell fragments which were cytoplasmic processes of agranulated folliculo-stellate cells were found. The cells showed polygonal irregular in shape and projected slender cytoplasmic processes between the non-reacted granulated cells. The S-100 protein is one of maker protein of the folliculo-stellate cell of the rat or human anterior pituitary. The reaction of S-100 protein showed that the reaction processed normally.
S-100 protein reaction X100
Figure 2-1: Immunohistochemistry of LH-RH reaction in the SC plane. Reactions were observed in the brain including the cephalad part of floor of the third ventricles. No reactions were observed in the pituitary area at this magnification. The infundibular stalk was connected to the pars nervosa and the floor. Around the ependymal layer, the reactions were observed. Blood vessels entered the brain. Rectangular part was schematically represented in Figure 2-2.
Anti-LH-RH stain X 20
Figure 2-2: A schematic representation of a sagittal section of the hypothalamus and the pars tuberalis of the pituitary gland. The pars tuberalis was tentatively separated into three portions for convenience of explanation from the cephalad to the caudal part. Primary plexus portion: PP. Cephalad half of the portal vein segment: APV. Caudal half of the portal vein segment: PPV. Similarly, the floor of the third ventricle was separated into three portions. Portion of the median eminence that corresponded to the primary plexus portion of pars tuberalis: PPM; anterior portion of the median eminence that corresponded to the portal vein segment of the pars tuberalis: APVM; caudal portion of median eminence corresponded to the portal vein segment of the pars tuberalis: PPVM. A small protuberance indicated by the arrow was named the transitional zone of the hypothalamus and infundibular recess (THI). A bend of the floor that was corresponded to the THI was named bend of the floor of the third ventricle (BFT). Arrowheads indicate the area expressed as "bottom of the floor" in the text.
Next 12 photographs (figures 3 to 6) are attached capital é“Lé” or é“Sé” following the figure number such as Figure 3L or Figure 3S. The é“Lé” indicates Immunohistochemistry of LH-RH and the é“Sé” indicates Immuno-histochemistry of S-100 protein.
Figure 3L: Immunohistochemistry of LH-RH on sagittal center sections. A few capillaries were attached to the é“bottom of the flooré” of the third ventricle. Some reaction end products were observed as dotted lines on the front portion of the floor of the third ventricle. A few arrays of deposits ran down to the capillaries, almost all of the dotted lines ran to PPVM. At the APVM and PPVM, arrays of deposits were observed just below the third ventricle and almost all of the lines were directed to the infundibular stalk. A few reaction end products were also observed from the THI to the infundibular stalk. Blood vessels were clearly observed between the brain tissue and the pars nervosa and vessels entering brain tissue were observed. Note disappearance of the PP of the pituitary gland in the SC plane. A short primary plexus (PP) attached to the é“bottom of the flooré”. The arrays of dots of anti-LH-RH reaction products ran on to the plexus at the PP area. A few arrays of the dots ran toward PPV. The dots of reaction of LH-RH increased in the THI. The BFT area was avascular.
X 100
Figure 3S: Immunohistochemistry of S-100 protein on sagittal center section. Anti-S-100 reactions in the brain at the SC were observed as usual. Only weakly positive cells were observed in the PPV. Intense reaction for S-100 was observed on the folliculo-stellate cells and their cell processes in the anterior lobe. Photographs of anti-S-100 reactions of the next three sagittal planes (100, 200 and 300um-SC) in higher magnifications showed findings with more clarity.
X 100
Figure 4L: At 100um-SC plane, the third ventricle was narrowed. Indicated area was a nonreaction area in the APVM.
X 100
Figure 4S: A few S 100 positive cells were observed in the pars tuberalis.
X 100
Figure 5L-1: At 200um-SC plane, numerous primary plexus were observed in the PP, which were not observed at the SC and 100um-SC. The arrays of dots of LH-RH reaction to the plexus at the PP area increased with increment of the plexus. It was very interesting though it was often on the PPVM that the arrays of deposits of anti-LH-RH reaction products ran to pars tuberalis.
X 100
Figure 5S-1: In contrast to the APV where few S-100 positive cells were seen, clusters of strongly S-100 positive cells were observed in the PPV area. The clusters showing the apical cell processes were also clearly observed.
X 100
Figure 5L-2: At the PPVM in this plane, the é“bottom of the flooré” approached and sometimes touched the PPV.
X 400
Figure 5S-2: High magnification of the rectangular part in figure 5S-1. S-100 positive cells were strongly observed in the PPV area. The clusters of cells showed interwinding cell processes.
X 400
Figures 5L-3 and -4: High magnification photographs at PPV. Arrows indicated slender LH-RH positive fibers in the pars tuberalis. In this area, the fiber was often observed. Arrowheads indicated the small round positive sites around the cells. Note the disappearance of a slender LH-RH fiber and a few varicosities at the cells.
X 400
Figure 6L: At 300um-SC plane, slender margin of third ventricle was observed. Only fragments of PP without primary plexus were observed. The array arrangements of LH-RH positive deposits on the APVM were increased. On the contrary to this observation for the APVM and APV, it was easy to find the arrangement within the PPV and the PPVM.
X 100
Figure 6S: At 300um-SC plane, a margin of third ventricle was observed. Few S-100 positive cells were observed.
X 100
Figure 7: Mirror images of Anti-LH-RH (7L) and S-100 (7S) stains 200um-SC. Mirror sections of the anti-LH-RH and S-100. Several round deposits of LH-RH reaction were observed (7L) around the S-100 positive cell (7S) in the PPV. Note the disappearance of a slender LH-RH fiber and a few varicosities (7L) around an S-100 positive cell (7S).
X 1,000
Figure 8-1: Non-myelinated nerve fibers (asterisk) and fragments of neuroendocrine cell in agranulated cells in PPV. The fragments stuffed numerous dense granules (arrow) (80-110 nm in diameter). The agranulated cells arranged around the small follicle (double arrow). Fragments of neuroendocrine cell in agranulated cells in PPV. The fragments contained dense granules (arrows). The dense granules were packed by membrane. No basement membrane was observed between the cell and the fragments. PP; Protrusion of the hypothalamus, PT; Pars tuberalis, NGC; Nongranulated folliculo-stellate cell
X 4,000
Figure 8-2: Fragments of neuroendocrine cell (asterisk) attached on agranulated cells at PPV. At PPV and PPVM, a small cell process contacted to an agranulated cell. The cell process contained small granules (80-110 nm in diameter). The cell process touched on the agranulated cell. The agranulated cells arranged around the small follicle (double arrow). PT: Pars tuberalis
X 16,000


Nagoya City University
Medical School